The present invention relates generally to the fields of matrices that are modified to enclose particularly designed fusion proteins. More particularly, it concerns the use of fusion proteins that include internal degradation sites and/or enzymatic cleavage sites. Artificial matrices may be designed having desired degradation rates, as well as to include particular active biological molecules, such as growth factor or enzyme.
It has been demonstrated that bi-domain peptides, which contain a factor XIIIa substrate sequence and a bioactive peptide sequence, can be cross-linked into fibrin gels and furthermore, that this bioactive peptide retains its cellular activity in vitro. While peptides can partially mimic the bioactivity of the whole protein from which they are derived, this bioactivity is usually lower than the bioactivity of the whole protein, and sometimes it is impossible to mimic certain proteins with only a short peptide. In order to incorporate the specific bioactivity of these type of factors, such as growth factors, it would be beneficial for the entire protein to be incorporated into the fibrin matrix.
Whole proteins can be incorporated into fibrin gels in a number of ways as taught in this invention. One method is to attach heparin to the gel by either covalent or non-covalent methods. This permits heparin-binding proteins including heparin-binding growth factors to be non-covalently bound to the fibrin gel. If the protein to be bound does not contain a native heparin-binding sequence, a fusion protein can be constructed containing the native protein sequence and a synthetic heparin-binding domain. Alternatively, a fusion protein can be constructed which contains a factor XIIIa substrate and the native protein sequence and this fusion protein can be sequestered by cross-linking it to the gel with factor XIIIa.
Fusion Protein Synthesis
Synthesis of either of the fusion proteins described above can be accomplished by utilizing molecular biology techniques. To do this, a fusion protein can be created that contains the entire protein sequence of interest with a cross-linking or binding sequence fused onto the amino or carboxyl terminus. This is done at the DNA level, as sequences encoding for either a factor XIIIa cross-linking substrate or a heparin-binding domain can be inserted at the beginning or the end of the codons for the original protein. When these modified proteins are expressed, they will then contain the additional domain of interest at the amino terminus. By using the natural machinery designed for protein synthesis, it becomes possible to synthesize and purify large proteins with high fidelity.
Incorporation of Fusion Proteins
Once the protein is purified, it can then be incorporated into the fibrin gels using several different schemes. In the first design, a factor XIIIa substrate has been directly incorporated onto the protein. When this modified protein is present during the polymerization of the fibrin, it is directly incorporated into the fibrin matrix in a manner similar to the bi-domain peptides previously demonstrated (14). A separate method involves fusion proteins that have been synthesized with a heparin-binding domain. In this example, a bi-domain peptide, heparin, and the heparin-binding fusion protein are included in the fibrin polymerization mixture. During polymerization, the bi-domain peptide is cross-linked into the fibrin gel. This bi-domain peptide would contain a factor XIIIa substrate sequence in addition to a heparin-binding sequence. The heparin binds to the bi-domain peptide that has been incorporated in the fibrin gel and is trapped in the fibrin matrix. This entrapped heparin serves to sequester the heparin-binding fusion protein within the fibrin gel by binding to the engineered heparin-binding domains. This incorporation has been shown to be stable enough to sequester the growth factor until the cross linked peptide is removed from the gel via cell controlled proteolysis.
This technique can be further modified by incorporating an enzymatic degradation site between the factor XIIIa substrate sequence and the sequence encoding the protein of interest. By careful selection of Km and kcat of this enzymatic degradation site, degradation could be controlled to occur either before or after the protein matrix and/or by utilizing similar or dissimilar enzymes to degrade the matrix, with the placement of the degradation site being tailored for each type of protein and application. This new protein could be directly cross-linked into the fibrin matrix as described above. However, incorporating an enzymatic degradation site alters the release of the protein during proteolysis. When the cell-derived proteases reach the sequestered protein, they can cleave the engineered protein at the newly formed degradation site. The resulting degradation products would include the liberated protein, which would now be free of any engineered fusion sequences, as well as any degraded fibrin. Therefore, the free protein would now be identical in primary sequence to the native growth factor and potentially more bioactive. A similar method can be used with the heparin-binding fusion proteins. These new proteins would then contain the protease degradation site, as well as the new heparin-binding domain. The heparin-binding fusion proteins will be sequestered into the matrix by the incorporation of heparin into the fibrin via the covalent immobilization of heparin-binding peptides. Once again, with the new protease degradation site added, the released protein would be identical in primary sequence to the natural protein.
Using standard molecular biology techniques, fusion proteins can be made of any growth factor for which the protein or DNA sequence is known, allowing the addition of novel domains such as heparin-binding domains or enzymatic substrates. These fusion proteins can be constructed so as to add a novel domain to either the N or C-terminus of the protein. The modifications are made at the DNA level by constructing a gene containing both the DNA sequence coding for the growth factor and the DNA sequence coding for a heparin-binding domain. This DNA is then ligated into an expression plasmid and transformed into bacteria. Upon induction of expression, the bacteria will produce large amounts of this fusion protein. Following expression, the protein must be purified from the cell lysate and refolded. Purification is often simplified due to the tendency of mammalian proteins expressed at high level to form inclusion bodies in bacteria.
The simplest way to incorporate proteins into fibrin is to attach heparin to the fibrin gels and use the heparin to sequester heparin-binding proteins, such as heparin-binding growth factors. This can be accomplished one of two ways, either by directly coupling a heparin-peptide chimera (where the heparin is chemically attached to a peptide containing a factor XIIIa substrate), or indirectly by cross-linking a heparin-binding peptide into the fibrin gel and binding heparin to this peptide non-covalently (using a bifunctional peptide containing a heparin-binding domain and a factor XIIIa substrate). This heparin can then sequester proteins, such as growth factors with heparin affinity, in the fibrin gel in a manner similar to the way that they are sequestered to the extracellular matrix in nature. Heparin can also protect these factors from proteolytic degradation and prolong their activity until they are released from the matrix.
Despite their relatively strong affinity for heparin, heparin-binding growth factors dissociate from the matrix on a short time scale. Therefore, a high excess of binding sites is essential to ensure that they do not diffuse far before they bind to the matrix again. This equilibrium also allows for the binding of free growth factor to cell surface receptors that are in close proximity to the site of dissociation. This method of controlled release provides both relatively long term binding of growth factors and rapid release of growth factors to local cells.
Heparin-binding domains naturally occur in many different families of growth factors. One of these families with one or more member that bind heparin are the fibroblast growth factors (13). Additional growth factors which bind heparin include transforming growth factor, interleukin-8, neurotrophin-6, vascular endothelial cell growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, connective tissue growth factor, midkine, and heparin-binding growth associated molecule (3, 7-10, 12, 16, 17, 20). These factors have shown the potential to enhance healing in many different types of tissue including vasculature, skin, nerve and liver. Therefore, these materials could be used to enhance wound healing in many different parts of the body by selecting the appropriate growth factor.
2. Approach 1: Heparin-binding Domain-factor XIIIa Substrate+Heparin to Attach Growth Factor
The attachment of heparin, either covalently or non-covalently to fibrin gels adds a novel functionality to these materials. The attachment of heparin permits the fibrin matrix to bind heparin-binding proteins, including growth factors in a manner which does not harm the protein, and prevents free diffusion of the protein from the gel. This allows for the controlled-release of heparin-binding proteins by one of two mechanisms, either degradation of the gel or binding of the protein to some other high affinity protein, such as a cell surface receptor.
Heparin can be attached to fibrin gels non-covalently using a two-part system consisting of a peptide chimera and heparin itself. The peptide chimera consists of two domains, a factor XIIIa substrate and a polysaccharide-binding domain. Once the peptide chimera is cross-linked into the fibrin gel, it attaches the heparin (or other polysaccharides) by non-covalent interactions.
Numerous proteins have been found to have heparin-binding affinity. Some of these proteins and the sequences of their heparin-binding domains are listed below.
Cross-linking Protocol for use of Heparin-Binding Peptides:
1) Dialyze fibrinogen (8 mg/ml) versus 4 L of Tris buffered saline (33 mM Tris), pH 7.4 for 24 hours.
2) Sterile filter fibrinogen using a 0.2 xcexcm syringe filter.
3) Make the following peptide solutions:
4) Make thrombin solution: 100 units in 5 ml TBS.
5) Add 1.4 ml of fibrinogen to each peptide solution.
6) Make gels: Add 20 xcexcl of TBS+50 mM CaCl2, 40 xcexcl of thrombin solution (20 units/ml), and 340 xcexcl of peptide solution+fibrinogen. (above solutions make 6 gels).
7) Incubate at 37C. for 1 hr.
8) Wash 5 times in 24 hours. Use 1 ml of TBS the first 4 times and neuronal media the last time.
9) Dissect day 8 chick embryonic dorsal root ganglia.
10) Place one ganglia in each gel and place at 37xc2x0 C. for 1 hr.
11) Add 1 ml of neuronal media to each gel.
12) Change media after 24 hours.
The results of these studies are shown in FIG. 2.
These results show that the heparin and peptide alone do not increase neurite extension. When added without peptide and heparin, bFGF does not enhance neurite outgrowth, demonstrating that the washing protocol used is sufficient. Neurite enhancement is increase by the addition of both 1 xcexcg/ml and 5 xcexcg/ml of bound bFGF in a dose dependent manner. The addition of 1.0 xcexcg/ml bound VEGF did not increase neurite extension, suggesting that the effect bFGF is not due to its ability to promote angiogenesis.
3. Approach 2: Polysaccharide Grafts (Heparinxe2x80x94Factor XIIIa Substrate Chimera) to Bind Growth Factor
Heparin (or other polysaccharides such as heparan sulfate of chondroitin sulfate) can be attached to fibrinogen directly using factor XIIIa by constructing a heparin-peptide chimera. This chimera contains two domains, a peptide domain consisting of a factor XIIIa substrate and the polysaccharide domain such as heparin. These chimeras are made using modified heparin (or another polysaccharide) which contains a unique reactive group at one end to control the site where coupling occurs on the heparin molecule. Though the use of a unique functional group on the peptide, such as a side chain present only on the end of the peptide where coupling is desired, the location of coupling on the peptide can be controlled as well. These chimeras can then be covalently cross-linked to fibrin gels using the sample methods as peptide chimeras, allowing direct attachment of heparin to the fibrin gel.